SUMMARY OF WORK: Project 2 ? Dissecting GRK Function in the Heart Research over the last two decades has shown that increased G protein-coupled receptor (GPCR) kinase-2 (GRK2) expression and activity in the cardiomyocyte, which can occur after stress/injury, is pathological to the heart. Studies have shown that lowering GRK2 expression or inhibiting its activity in myocytes rescues several different animal models of heart failure (HF). Mechanistically, new data has revealed that GRK2-mediated myocardial pathology can occur through both GPCR-dependent and GPCR-independent actions. Over the last funding cycle of this PPG, we found, using genetically engineered mice including a novel GRK2 knock-in mouse (GRK2-C340S) where endogenous GRK2 can no longer be regulated via nitric oxide (NO) bioavailability (through S-nitrosylation), that GRK2 can induce cardiac injury through disruption of the normal nitroso-redox balance of the myocyte. More recent data demonstrates that increased GRK2 can induce cellular reactive oxygen species (ROS) and this appears to be mitochondrial in origin, which is interesting since we have discovered GRK2 can reside in mitochondria. In this competitive renewal we are interested in delineating the mechanistic role of GRK2 on the oxidative state and nitroso-redox imbalance of the injured and stressed cardiomyoycte, especially during HF development. Importantly, since other components of GPCR signaling are also targets of S-nitrosylation, we have the unique opportunity through this PPG group to dissect GRK2/?-arrestin/GPCR mechanisms when NO bioavailability is altered. Finally, we have recently uncovered that the oxidative stress induced in the heart by the neurohormone aldosterone, which is implicated in human HF development through activation of its mineralocorticoid receptor, can also involve GRK2 and this will be explored herein. This project will specifically test the overall hypothesis that GRK2 is a key component of myocyte oxidative stress imbalance including through its inter-relationship with the NO signaling system and mitochondrial targeting. Moreover, we believe that GRK2 plays a key and novel role in promoting myocyte injury downstream of hyper-aldosteronism. These studies will also delineate new mechanisms supporting the therapeutic benefits of GRK2 lowering and inhibition in dysfunctional myocardium. Specific Aims are: [1] To determine whether dynamic S-nitrosylation plays a role in GRK2-mediated pathology during HF development; [2] To determine the mitochondrial targets of GRK2 in the myocyte that play a mechanistic role in altering mitochondrial function including metabolism and ROS formation; and [3] To determine the role of GRK2 in aldosterone-mediated cardiac pathology.